Bacteriophage SPO2-mediated plasmid transduction in transpositional mutagenesis within the genus Bacillus.

A single copy of the Streptococcus faecalis transposon Tn917, located in the Bacillus subtilis chromosome, was able to transpose onto the SPO2 cos plasmid pPL1017, which codes for chloramphenicol resistance and contains the bacteriophage phi 105 immunity region. Selection for pPL1017::Tn917 chimeras was performed by SPO2-mediated plasmid transduction of transposon-borne resistance to macrolide-lincosamide-streptogramin B antibiotics (MLSr). The transposition of Tn917 onto plasmid pPL1017 occurred with a frequency of 10(-5) and was dependent on the presence of a subinhibitory dose of erythromycin. Twelve chimeras were subjected to genetic and physical analyses. Two Cams transductants harbored plasmids whose chloramphenicol acetyltransferase genes had been insertionally inactivated by Tn917. Several transpositions in the vicinity of the phi 105 immunity region were detected. However, all of the 300 MLSr, Camr transductants screened were immune to phi 105 infectious activity. One pPL1017::Tn917 chimera, pLK200, was transferred by SPO2 plasmid transduction into the Bacillus amyloliquefaciens prototrophic strain DSM7. Plasmid pLK200 was effective in the mutagenesis of the DSM7 chromosome and yielded auxotrophs at a frequency of 0.5 to 5.3%. Generation of auxotrophs was also dependent on the presence of a subinhibitory dose of erythromycin. Forty-four auxotrophs representing at least nine amino acid requirements were recovered.     

Brucella abortus in captive bison. I. Serology, bacteriology, pathogenesis, and transmission to cattle

Two groups of six, non-brucellosis vaccinated, brucellosis seronegative pregnant American bison (Bison bison) were individually challenged with 1 x 10(7) colony forming units (CFU) of Brucella abortus strain 2308. Three days after challenge, each bison group was placed in a common paddock with six non-vaccinated, brucellosis susceptible, pregnant domestic heifers. In a parallel study, two groups of six susceptible, pregnant cattle were simultaneously challenged with the identical dose as the bison and each group was placed with six susceptible cattle in order to compare bison to cattle transmission to that observed in cattle to cattle transmission. Blood samples were collected from bison and cattle weekly for at least 1 mo prior to exposure to B. abortus and for 180 days post-exposure (PE). Sera from the bison and cattle were evaluated by the Card, rivanol precipitation, standard plate agglutination, standard tube agglutination, cold complement fixation tube, warm complement fixation tube, buffered acidified plate antigen, rapid screening, bovine conjugated enzyme linked immunosorbent assay, bison or bovine conjugated enzyme linked immunosorbent assay, and the hemolysis-in-gel techniques for the presence of antibodies to Brucella spp. At the termination of pregnancy by abortion or birth of a live-calf, quarter milk samples, vaginal swabs, and placenta were collected from the dam. Rectal swabs were collected from live calves, and mediastinal lymph nodes, abomasal contents and lung were taken at necropsy from aborted fetuses for culture of Brucella spp. These tissues and swabs were cultured on restrictive media for the isolation and identification of Brucella spp. Pathogenesis of brucellosis in bison was studied in an additional group of six pregnant bison which were challenged with 1 x 10(7) CFU of B. abortus strain 2308. One animal was euthanatized each week PE. Tissues were collected at necropsy and later examined bacteriologically and histologically. Lesions of brucellosis in bison did not significantly differ grossly or histologically from those in cattle. There were six abortions and two nonviable calves in the bison group, as compared to nine abortions in the 12 similarly inoculated cattle. As determined by bacterial isolations, transmission of B. abortus from bison to cattle (five of 12 susceptible cattle became infected) did not differ statistically from cattle to cattle transmission (six of 12 susceptible cattle became infected) under identical conditions. No single serologic test was constantly reliable to diagnosing B. abortus infected bison for 8 wk PE.(ABSTRACT TRUNCATED AT 400 WORDS)     

High-throughput fluorescent tagging of full-length Arabidopsis gene products in planta

We developed a high-throughput methodology, termed fluorescent tagging of full-length proteins (FTFLP), to analyze expression patterns and subcellular localization of Arabidopsis gene products in planta. Determination of these parameters is a logical first step in functional characterization of the approximately one-third of all known Arabidopsis genes that encode novel proteins of unknown function. Our FTFLP-based approach offers two significant advantages: first, it produces internally-tagged full-length proteins that are likely to exhibit native intracellular localization, and second, it yields information about the tissue specificity of gene expression by the use of native promoters. To demonstrate how FTFLP may be used for characterization of the Arabidopsis proteome, we tagged a series of known proteins with diverse subcellular targeting patterns as well as several proteins with unknown function and unassigned subcellular localization.     

Alzheimer's β-Amyloid Peptide 1–42 Induces a Phagocytic Response in Murine Microglia

Abstract: β-Amyloid (Aβ) peptides are a key component of the senile plaques that characterize Alzheimer's disease. Cytokine-producing microglia have been shown to be intimately associated with amyloid deposits and have also been implicated as scavengers responsible for clearing Aβ deposits. However, little is known about the initial activation of these microglia or the effect of Aβ on phagocytosis. Murine BV-2 microglia were used to assess the effect of synthetic Aβ 1–42 on phagocytosis by quantifying uptake of fluorescent microspheres, acetylated low-density lipoproteins, and zymosan particles by flow cytometry. Aβ 1–42 stimulated microglial phagocytosis in a time- and dose-dependent manner. Aβ fibrils produced the greatest potentiation, and once activated, phagocytosis remained elevated after removal of Aβ from the cultures. Aβ-stimulated phagocytosis could be blocked if proteoglycans were first complexed to Aβ fibrils. These data suggest that Aβ fibrils act as an immune signal to stimulate microglial phagocytosis and that extracellular matrix molecules may modify Aβ function.     

Genetic engineering of the major timothy grass pollen allergen, Phl p 6, to reduce allergenic activity and preserve immunogenicity

On the basis of IgE epitope mapping data, we have produced three allergen fragments comprising aa 1-33, 1-57, and 31-110 of the major timothy grass pollen allergen Phl p 6 aa 1-110 by expression in Escherichia coli and chemical synthesis. Circular dichroism analysis showed that the purified fragments lack the typical alpha-helical fold of the complete allergen. Superposition of the sequences of the fragments onto the three-dimensional allergen structure indicated that the removal of only one of the four helices had led to the destabilization of the alpha helical structure of Phl p 6. The lack of structural fold was accompanied by a strong reduction of IgE reactivity and allergenic activity of the three fragments as determined by basophil histamine release in allergic patients. Each of the three Phl p 6 fragments adsorbed to CFA induced Phl p 6-specific IgG Abs in rabbits. However, immunization of mice with fragments adsorbed to an adjuvant allowed for human use (AluGel-S) showed that only the Phl p 6 aa 31-110 induced Phl p 6-specific IgG Abs. Anti-Phl p 6 IgG Abs induced by vaccination with Phl p 6 aa 31-110 inhibited patients' IgE reactivity to the wild-type allergen as well as Phl p 6-induced basophil degranulation. Our results are of importance for the design of hypoallergenic allergy vaccines. They show that it has to be demonstrated that the hypoallergenic derivative induces a robust IgG response in a formulation that can be used in allergic patients.     

A Mediterranean-style low-glycemic-load diet increases plasma carotenoids and decreases LDL oxidation in women with metabolic syndrome

Thirty-five women with metabolic syndrome and high plasma low-density lipoprotein (LDL) cholesterol (≥100 mg/dl) participated in a dietary intervention consisting of a Mediterranean-style low-glycemic-load diet for 12 weeks. Participants were randomly allocated to consume diet only (n=15) or diet plus a medical food containing soy protein and plant sterols (n=20). Plasma concentrations of carotenoids, lipoprotein subfractions and oxidized LDL (OxLDL) were measured. Independent of treatment, women had a significant increase in plasma lutein (P<.0001) and β-carotene (P<.0001), while plasma lycopene was reduced (P<.05) after 12 weeks. Low-density lipoprotein cholesterol was reduced from 138±35 to 114±33 mg/dl (P<.0001). In addition, decreases were observed in the atherogenic subfractions: large very low-density lipoprotein (P<.05), small LDL (P<.00001) and medium high-density lipoprotein (P<.05). Oxidized LDL was significantly reduced by 12% in both groups (P<.01). Changes in OxLDL were inversely correlated with plasma lutein (r=-.478, P<.0001). The data indicate that women complied with the dietary regimen by increasing fruits and vegetable intake. Decreased consumption of high-glycemic foods frequently co-consumed with lycopene-rich tomato sauce such as pasta and pizza may be responsible for the lowering of this carotenoid in plasma after 12 weeks. These results also suggest that plasma lutein concentrations may protect against oxidative stress by reducing the concentrations of OxLDL.     

WAFL, a new protein involved in regulation of early endocytic transport at the intersection of actin and microtubule dynamics

We have previously identified a new gene with sequence homology to the WASP-family of actin regulators denoted WAFL (WASP and FKBP-like). Here we report a possible biological function for WAFL, by demonstrating an association to early endosomes via its central coiled-coil domain. Further we show by functional and structural studies that WAFL is associated with both microtubules and the actin filament system, the two means of transport of early endosomes. In addition, WAFL interacts with WASP-interacting protein (WIP) and actin, thus linking WAFL to actin dynamics. The use of RNAi depletion of WAFL shows that WAFL-deficient cells display delayed transport of endosomal cargo. Our findings are compatible with a model whereby WAFL is involved in the transport of early endosomes at the level of transition between microfilament-based and microtubule-based movement.